Ischemic heart disease is the main cause of morbidity and mortality. The epidemiological and socio-economical impact of coronary heart disease is remarkable. This disease causes million of deaths all over the world. See Murray, et al., Lancet, 349:1269-1276 (1997). In developed countries, it has been estimated that 5.3 million deaths attributable to cardiovascular disease occurred in 1990, whereas the corresponding figure for the developing countries ranged between 8 to 9 million (showing a relative excess of 70%). See Reddy, et al., Circulation, 97:596-601 (1998). In Argentina, ischemic heart disease is the first cause of mortality showing an incidence of around 30%, trend which tends to remain stable since 1980. For the population over 65 years, this rate reaches almost 40%. See Programa Nacional de Estadisticas de Salud, Series 5, Number 38, Ministerio de Salud y Acción Social, República Argentina (December 1995).
Despite recent advances in prevention and treatment of ischemic heart disease, there are many patients who are still symptomatic and cannot benefit from conventional therapy. Administration of growth factors that promote neovascular formation and growth, such as fibroblast growth factors (FGFs) and VEGF, appear as a novel and promising alternative for these patients. This mode of treatment is called therapeutic angiogenesis. See Henry, B. M. J., 318:1536-1539 (1999).
VEGF is a protein expressed by skeletal muscle cells, smooth muscle cells, ovarian corpus luteum cells, tumor cells, fibroblasts and cardiomyocytes. Unlike other mitogens, VEGF is a secreted growth factor. See Thomas, J. Biol. Chem, 271:603-606 (1996); Leung, et al., Science, 246:1306-1309 (1989). The human VEGF gene is expressed as different isoforms, secondary to post-transcriptional alternative splicing. In non-malignant human tissues, four VEGF isoforms are expressed, with different numbers of amino acids (121, 165, 189, 206) and with a molecular weight ranging from 34 to 46 kD. See Tischer, et al., J. Biol. Chem., 266:11947-11954 (1991); Ferrara, et al., J. Cell. Biochem., 47:211-218 (1991).
VEGF specific receptors are VEGFR-1 (flt-1), VEGFR-2 (KDR/flk-1) and VEGFR-3 (flt-4). See De Vries, et al., Science, 255:989-991 (1992); Terman, et al., Biochem. Biophys. Res. Commun., 187:1579-1586 (1992); Gallant, et al., Genomics, 13:475-478 (1992). Due to the apparent restricted and confined localization of VEGF receptors to vascular endothelial cells, this growth factor has been described as the most specific mitogen for these cells. It has been proposed that VEGF is not bioactive on non-endothelial cells. See Jakeman, et al., J. Clin. Invest., 89:244-253 (1992); Ferrara, et al., Endocr. Rev., 18:4-25 (1997); Thomas, et al., supra (1996). However, recent studies have reported mitogenic effects of VEGF on some non-endothelial cell types, such as retinal pigment epithelial cells, pancreatic duct cells and Schwann cells. See Guerring et al., J. Cell. Physiol., 164:385-394 (1995); Oberg-Welsh et al., Mol. Cell. Endocrinol., 126:125-132 (1997); Sondell et al., J. Neurosci., 19:5731-5740 (1999). Moreover, VEGF receptors have been found in other cells, such as hematopoietic stem cells, endocardial cells and even cultured rat cardiomyocytes, where VEGF has been shown to activate the mitogen-activated protein kinase cascade. See Asahara et al., Science, 275:964-967 (1997); Partanen et al., Circulation, 100:583-586 (1999); Takahashi et al., Circ. Res., 84:1194-1202 (1999).
Therapeutic administration of VEGF is a significant challenge. VEGF can be administered as a recombinant protein (protein therapy) or by VEGF-encoding gene transfer (gene therapy). See Safi, et al., J. Mol. Cell. Cardiol., 29:2311-2325 (1997); Simons, et al., Circulation, 102:E73-E86 (2000).
Protein therapy has several disadvantages. The extremely short mean-life of angiogenic proteins (e.g. VEGF) conditions therapy to the administration of high or repeated doses to achieve a noticeable effect. See Simons, et al., supra (2000); Takeshita, et al., Circulation, 90:II228-234 (1994). Furthermore, intravenous administration of high doses of VEGF protein is known to induce severe or refractory hypotension. See Henry, et al, J. Am. Coll. Cardiol., 31:65A (1998); Horowitz, et al., Arterioscl. Thromb. Vasc. Biol., 17:2793-2800 (1997); López, et al., Am. J. Phisiol., 273:H1317-1323 (1997). To avoid these disadvantages, gene therapy (e.g. DNA encoding for VEGF) has been proposed. See Mack, et al., J. Thorac. Cardiovasc. Surg., 115:168-177 (1998); Tio, et al., Hum. Gene Ther., 10:2953-2960 (1999).
Gene therapy can be compared to a drug slow-delivery system. The gene encoding for the agent of interest is transported into cells in vehicles called vectors (e.g. plasmids, viruses, liposomes). Cell mechanisms specialized in protein synthesis perform the production and localized release of the final product. See Crystal, Science, 270:404-410 (1995). In addition, it should be noted that in the case of plasmids the gene product is synthesized for a discrete period of time. This time is usually about two weeks. According to experimental studies, sustained expression during this limited period of time is necessary and sufficient to trigger the angiogenic process. Based on these advantages, several research groups have studied the therapeutic effects of gene therapy using angiogenic factors in experimental models of heart and limb ischemia. These approaches have yielded promising results. See Magovern, Ann. Thorac. Surg., 62:425-434 (1996); Mack, et al., supra (1998); Tio, et al., supra (1999); Walder, et al., J. Cardiovasc. Pharmacol., 27:91-98 (1996); Takeshita, et al., Lab. Invest., 75:487-501 (1996); Mack, et al., J. Vasc. Surg., 27:699-709 (1998); Tsurumi, et al., Circulation, 94:3281-3290 (1996). Gene therapy has achieved the expected effects without the shortcomings associated with protein therapy. However, adenoviral gene therapy may induce inflammatory or immune reactions, especially after repeated doses. This type of therapy has been related also to high risk systemic immune response syndrome. These circumstances limit significantly the clinical use of this therapy. See Gilgenkrantz, et al., Hum. Gene Ther., 6:1265-1274 (1995); Dewey, et al., Nat. Med., 5:1256-1263 (1999); Wersto, et al., J. Virol., 72:9491-9502 (1998); Hollon, Nat. Med., 6:6 (2000), Chan, et al., Nat. Med., 5:1143-1149 (1999); Byrnes, et al., J. Nerosci., 16:3045-3055 (1996). According to recent studies, plasmid gene therapy does not have these disadvantages and can be administrated safely in repeated doses. See Simons, et al., supra (2000).
Systemic administration of VEGF has been associated with undesired angiogenesis in peripheral tissues. See Folkman, Nat. Med., 1:27-31 (1995); Liotta, et al., Cell, 64:327-336 (1991); Lazarous, et al., Circulation, 94:1074-1082 (1996); Ferrara, Breast Cancer Res. Treat., 36:127-137 (1995); Ferrara, Lab. Invest., 72:615-618 (1995); Aiello, et al., N. Eng. J. Med., 331:1480-1485 (1994); Adams, et al., Am. J. Ophthalmol., 118:445-450 (1994); Inoue, et al., Circulation, 98:2108-2116 (1998); Simons, et al., supra (2000). The risk of systemic exposure is probably more related to the route of administration than to the nature of therapy (gene or protein) utilized. In comparison with intravascular delivery, local (e.g. intramyocardial) administration reduces the risk of systemic exposure and undesired peripheral angiogenesis. See Simons, et al., supra (2000).
At the present, it has been demonstrated that VEGF induces angiogenesis in vivo. It has not been reported yet that VEGF induces the formation of blood vessels with a smooth muscle layer. See Mack, et al., supra (1998); Tio, et al., supra (1999). Moreover, it has been postulated that VEGF prevents the neoformation of vascular smooth muscle. See Asahara, et al., Circulation, 91:2793-2801 (1995). Smooth muscle plays a significant role in the regulation of vascular function. Its presence at the media layer of blood vessels represents an adaptative advantage since it is involved in the vasomotor tone regulation. Vascular smooth muscle maintains a basal vascular tone and permits self-regulation upon variations on blood flow and pressure. It has been suggested that the absence of smooth muscle layer is related to vessel collapse. See “Angiogenesis and Cardiovascular Disease”, Ware, Ed. (Oxford University Press Inc., New York, USA., 1999), p. 258-261.
Acute myocardial infarction is the consequence of coronary heart disease with the worst short and long-term prognosis. See Bolognese, et al., Am. Heart J., 138:S79-83 (1999); Mehta, et al., Herz, 25:47-60 (2000); Hessen, et al., Cardiovasc. Clin., 20:283-318 (1989); Jacoby, et al., J. Am. Coll. Cardiol., 20:736-744 (1992); Rosenthal, et al., Am. Heart J., 109:865-876 (1985). This condition results frequently in a significant loss of myocardial cells, reducing the contractile muscle mass. It is known in the art that cardiomyocytes of human and human-like species preserve their ability to replicate DNA. See Pfizer, et al., Curr. Top. Pathol., 54:125-168 (1971). Recently, it has been informed that some human cardiomyocytes can enter into M (mitotic) phase. However, this phenomenon occurs in a very small proportion of total cardiomyocyte population and under certain pathological conditions. So far, this phenomenon has only been noted in myocardial infarction and end-stage cardiac failure. See Beltrami et al., N. Eng. J. Med., 344: 1750-1757 (2001); Kajstura, et al., Proc. Natl. Acad. Sci. USA, 95:8801-8805 (1998). There is no conclusive evidence in all these instances that cardiomyocytes divide into daughter cells.
The inability of cardiomyocytes to replicate properly precludes the replacement of myocardial tissue after injury in upper animal species. Under this scenario, myocardial function is diminished because the infarcted area is replaced by fibrotic tissue without contractile capacity. In addition, the remaining cardiomyocytes become hypertrophic and develop polyploid nuclei. See Herget, et al., Cardiovasc. Res. 36:45-51 (1997); “Textbook of Medical Physiology”, 9th Ed., Guyton et al., Eds. (W. B. Saunders Co, USA, 1997).
Attempts have been made to restore myocardial cell loss with other cells, such as autologous satellite cells and allogenic myoblasts. The results of these attempts are not conclusive. See Dorfman, et al., J. Thorac. Cardiovasc. Surg., 116:744-751 (1998); Murry, et al., J. Clin. Invest., 98: 2512-2523 (1996); Leor, et al., Circulation, 94 Suppl.II: II-332-II-336 (1996); Li et al., Circ. Res., 78:283-288 (1996). More recently, it has been suggested that pluripotent stem cells and bone marrow derived angioblasts might restore infarcted myocardial tissue and induce even neovascular formation. However, the efficiency of these methods in upper mammals has not been demonstrated yet. See Orlic, et al., Nature, 410:701-705 (2001); Kocher, et al., Nat. Med., 7:430-436 (2001). An ideal method should induce cardiomyocyte division originating daughter cells and neovascular formation in myocardial tissue. This procedure would restore tissue loss with autologous myocardial tissue and increase simultaneously myocardial perfusion. A method like this would reduce the morbility and mortality rates associated to left ventricular remodeling, myocardial infarction and ischemic heart disease. See Bolognese, et al., supra (1999).
Likewise, the failure of cardiomyocytes to replicate properly difficult adaptative hyperplasia (i.e. cell number increasing) as a response to other pathological conditions. In these cases, the adaptative response of human and porcine cardiomyocytes is to increase cell volume and nuclear DNA content. Therefore, in certain pathologies (e.g. hypertensive heart disease, dilated cardiomyopathy) cardiomyocytes are also markedly hypertrophic and polyploid. See Pfizer, Curr. Top. Pathol., 54:125-168 (1971); Adler, et al., J. Mol. Cell. Cardiol., 18:39-53 (1986). In most cases, cell adaptation is insufficient. Besides, the cellular demand for oxygen and nutrients increases as myocardial hypertrophy progresses. In consequence, the increased demands impair subendocardial perfusion even in the absence of coronary occlusion. Finally, the combination of these factors leads to myocardial function detriment. See “Textbook of Medical Physiology”, 9th Ed, supra. An ideal method should induce mitosis on hypertrophic and polyploid cells. This method should result in smaller and better-perfused daughter cells thus reducing the progression of cardiomyopathy towards heart failure.